The effects of mitogens on cell proliferation are mediated by increases in both transcription and translation. Much is known about the signaling pathways that link extracellular signals to the nucleus, but less is known about the precise mechanisms involved in the regulation of protein synthesis. Recent studies by a number of investigators have shown that protein translation is regulated by a signaling pathway that is inhibited by the immunosuppressant, rapamycin. Rapamycin in a complex with its binding protein, FKBP-12, binds to a protein termed FRAP (FKBP12-rapamycin-associated protein). By an unknown mechanism the binding of FKBP12-rapamycin to FRAP interferes with signaling initiated by receptor activation of phosphatidylinositol-3-kinase (PI3K). The most well characterized effects of rapamycin are inhibition of p70S6 kinase (p70S6K), a regulator of translational initiation, and decreased phosphorylation of 4E-BP1, a regulator of initiation factor eIF-4E. FRAP is related in primary amino acid sequence to the family of lipid kinases that includes PI3K, however, it is thought to be a protein kinase. Potential substrates for FRAP are 4E-BP1 and regulators of protein phosphatase activity. This application is aimed at investigating several aspects of FRAP. In Aim I, the function of FRAP will be investigated through the identification of novel substrates. The relationship of these substrates to the regulation of p70S6K and 4E- BP1 will be studied. In Aim II, the role of the domain of FRAP that binds FKBP12-rapamycin will be studied. In Aim III, novel proteins that associate with FRAP in a multi-protein complex will be purified and characterized. In Aim IV, the relationship of FRAP to the regulation of protein phosphatases will be studied. By understanding the detailed biochemical properties of FRAP, fundamental questions of how protein translation is coupled to cell cycle progression will be answered.